嗜水气单胞菌S蛋白的提纯及特性分析

PURIFICATION AND CHARACTERIZATION OF S-LAYER PROTEIN FROM AEROMONAS HYDROPHILA

电镜观察表明,嗜水气单胞菌J-1株具有S层结构,在菌体外层呈晶格样规则排列。菌体经酸性甘氨酸缓冲液处理,S层从菌体上脱落,离心上清液即为粗提S蛋白。进一步经Sephadex G200凝胶层析和DEAE-纤维素离子交换层析纯化,获得的S蛋白呈单一多肽,分子量为51500。氨基酸组分分析结果表明,S蛋白含有天门冬氨酸等15种氨基酸,其中丙氨酸等疏水性氨基酸占36.8％。生物学活性显示,S蛋白对Vero细胞有轻微的细胞毒性,但没有溶血性,对鲫鱼和小鼠也无致死作用。用自制的嗜水气单胞菌J-1株S蛋白抗血清PM及PR和国外提供的嗜水气单胞菌TF7株S蛋白抗血清PF1分别作免疫转印和间接ELISA,检测来源于不同地区和不同动物种类的20株嗜水气单胞菌的S蛋白。结果表明,S蛋白的抗原性存在着菌株间的差异。另外,某些菌株不具有S层。

The regular surface protein array (S-layer) present on Aeromonas hydrophila J-l was composed of a single species of protein of apparent molecular weight 51500. This protein was extracted from whole cells by treatment with 0.2mol / L glycine hydrocholoide (pH4.0). The protein was purified by Sephadex G-200 gel filtration chromatography and an ion exchange chromatography on DEAE-cellulose. Amino acid composition analysis showed, that the protein contained 36.8% hydrophobic amino acids. But the protein did not confer hydrophobicity to the cell surface when present as an intact S-layer by salt aggregation test. ELISA and immunoblotting with two different polyclonal antisera against surface exposed-(PM)and non-surface-exposed epitopes (PR) of the protein revealed that the sensitivity of PM was higher than that of PR. Antigenic diversity of the S-layer proteins from 20 bacterial samples was analysed by ELISA with PM and PF1 (polyclonal antiserum aganist Aeromonas hydrophila TF7 S-layer protein) . The S-layer proteins were distinguishable from the extracellular toxin of the homogeneous strains in antigenic and biochemical characterization and the S-layer proteins were one of the main protective antigens.